Abstract

OBJECTIVE Long-chain n-3 polyunsaturated fatty acid (n-3 PUFA) supplements may have renoprotective effects in patients with diabetes,
but previous trials have been inconsistent. We performed a randomized controlled trial of n-3 PUFA supplementation on urine
albumin excretion and markers of kidney injury in adults with type 2 diabetes.

RESEARCH DESIGN AND METHODS We conducted a randomized, placebo-controlled, two-period crossover trial to test the effects of 4 g/day of n-3 PUFA supplementation
on markers of glomerular filtration and kidney injury in adults with adult-onset diabetes and greater than or equal to trace
amounts of proteinuria. Each period lasted 6 weeks and was separated by a 2-week washout. The main outcome was urine albumin
excretion and, secondarily, markers of kidney injury (kidney injury molecule-1, N-acetyl β-d-glucosaminidase [NAG], neutrophil gelatinase-associated lipocalin [NGAL], and liver fatty acid–binding protein [LFABP]),
serum markers of kidney function (cystatin C, β2-microglobulin, and creatinine), and estimated glomerular filtration rate (eGFR).

RESULTS Of the 31 participants, 29 finished both periods. A total of 55% were male, and 61% were African American; mean age was 67
years. At baseline, mean BMI was 31.6 kg/m2, median eGFR was 76.9 mL/min/1.73 m2, and median 24-h urine albumin excretion was 161 mg/day. Compared with placebo, n-3 PUFA had nonsignificant effects on urine
albumin excretion (−7.2%; 95% CI −20.6 to 8.5; P = 0.35) and significant effects on urine NGAL excretion (−16% [−29.1 to −0.5%]; P = 0.04). There was no effect on serum markers of kidney function or eGFR. In subgroup analyses, there were significant decreases
in 24-h urinary excretion of albumin, NGAL, LFABP, and NAG among participants taking medications that block the renin-angiotensin-aldosterone
system (RAAS).

CONCLUSIONS These results suggest a potential effect of n-3 PUFA supplementation on markers of kidney injury in patients with diabetes
and early evidence of kidney disease. In the context of prior studies, these results provide a strong rationale for long-term
trials of n-3 PUFA on chronic kidney disease progression.

Diabetes is a leading cause of chronic kidney disease (CKD) (1). Treatments to slow the progression of CKD in diabetes include blocking the renin-angiotensin-aldosterone system (RAAS),
implementing lower blood pressure (BP) treatment goals, and treating hyperglycemia (2). These therapies can also reduce urine protein excretion, a marker of disease severity. Indeed, maximal reduction of urine
protein excretion has been proposed as a goal of drug therapy (3). Long-chain n-3 polyunsaturated fatty acid (n-3 PUFA) supplements may improve endothelial function, lower BP, and have independent
antiproteinuric effects (4). However, evidence of benefit from supplementation with n-3 PUFA on urine protein excretion in the setting of diabetic kidney
disease is inconsistent (5–11).

New markers of kidney function and injury hold considerable promise as a means to evaluate the potential benefits of therapies
designed to retard the progression of CKD. Biomarkers of tubulointerstitial kidney damage, including kidney injury molecule-1
(KIM-1), N-acetyl β-d-glucosaminidase (NAG), neutrophil gelatinase-associated lipocalin (NGAL), and liver fatty acid–binding protein (LFABP) may
have greater sensitivity for identifying effects on kidney injury than total urine protein or albumin excretion, which reflect
both kidney injury and hemodynamic effects (12,13). Novel serum markers, including β2-microglobulin and cystatin C, may provide greater sensitivity for determining short-term effects of therapies on estimated
glomerular filtration rate (eGFR) than traditional markers of filtration. These new markers of kidney function and injury
might be especially useful in guiding the design of subsequent long-term trials.

In this context, we conducted a randomized, controlled crossover trial to evaluate the efficacy of n-3 PUFA supplements on
improving markers of kidney injury and function in adults with adult-onset diabetes and greater than or equal to trace amounts
of proteinuria.

Inclusion criteria were a self-reported diagnosis of diabetes, either treated with oral medication(s) or diet-controlled;
age >21 years; an average systolic BP (SBP) <150 mmHg and diastolic BP (DBP) <90 mmHg during two screening visits; and a quantified
level of proteinuria greater than or equal to trace but <4+ on urine dipstick analysis during screening. If participants were
taking antihypertensive, hypoglycemic, or lipid-lowering medications, we required stable doses for a minimum of 2 months prior
to randomization. Exclusion criteria were poorly controlled diabetes (self-reported hemoglobin A1c [HbA1c] >9%); use of insulin; being pregnant or planning on becoming pregnant during the study period; unwillingness to stop taking
fish oil supplements 1 month prior to randomization for those on fish oil supplements; or an eGFR <30 mL/min (stage 4 or stage
5 CKD) based on the Modification of Diet in Renal Disease equation (14). The Johns Hopkins Institutional Review Boards approved the study protocol. All participants provided written informed consent.

Randomization and interventions

If participants were eligible based on their screening visits, they were asked to complete a 2–4-week run-in period during
which they took four corn oil capsules per day. After run-in, participants who remained interested and eligible were randomized.
Randomized assignments were generated from a random-numbers table, placed in sealed opaque envelopes, and opened by the study
coordinator in consecutive order. An 8-week supply of capsules was provided to participants in preallocated monthly pill packs.
Participants, all study staff, and analytic team members were masked to the randomization sequence.

Participants took four capsules per day during the intervention period. Each 1-g capsule of n-3 PUFA contained ∼85% n-3 fatty
acid ethyl esters sourced from fish oils, a combination of ethyl esters of eicosapentaenoic acid (EPA) and docosahexaenoic
acid (DHA) in an ∼2:1 ratio. A 2-week washout separated the two periods. The placebo capsules were identical in appearance
to the n-3 PUFA capsules and contained an isocaloric equivalent of corn oil. Pharmacokinetic studies suggested that a 6-week
period would be adequate for serum levels of n-3 PUFA to reach steady state and that a 4-week period would provide sufficient
time for serum levels to return to baseline levels following withdrawal (15,16). Participants were asked not to alter medication regimens of drugs that affect BP, diabetes, or lipids.

Data collection

Study participants completed visits at baseline and at the end of each treatment period. Weight, height, and waist circumference
were measured. BP was measured by the oscillometric technique using an OMRON 907 machine (Omron, Vernon Hills, IL). A set
of three readings (separated by 30 s) after 5 min of rest was performed following a standardized protocol.

Study participants provided fasting blood samples and 24-h urine collections at each of the three clinic visits. Blood samples
were allowed to clot at room temperature for 15 min and centrifuged at 2°C. Serum aliquots were stored at −70°C. Aliquots
of 24-h urine collections were collected and stored at −70°C. Fasting serum levels of total cholesterol, HDL cholesterol,
triglycerides, fasting serum glucose, and liver function tests were measured by a local laboratory (Quest Diagnostics). LDL
cholesterol was estimated using the Friedewald equation (17). Erythrocyte fatty acids including DHA and EPA were isolated by solid-phase extraction, identified, and quantitated by gas
liquid chromatography–mass spectrometry in the Peroxisome Diseases section of the Kennedy Krieger Institute using standard
methodologies (18).

Study outcomes

The primary outcome variable was 24-h urine albumin excretion. Secondary outcome variables were eGFR, cystatin C, and β2-microglobulin measured in fasting serum and NAG, LFABP, NGAL, and KIM-1 measured in 24-h urine samples. All outcome variables
were measured at baseline and the end of each treatment period. Laboratory assays of outcome measures were performed at the
Cincinnati Children’s Hospital Medical Center by personnel who were masked to participant randomized sequence. Urine albumin
was measured by immunoturbidometry using Dimension Xpand Plus clinical chemistry system (Siemens). Serum cystatin C and β2-microglobulin were measured by particle-enhanced immunonephelometric assays (Dade Behring, Deerfield, IL). Urine NAG activity
was measured using a colorimetric assay (Roche Diagnostics) as previously described (19). Urine NGAL was assayed using a human-specific commercially available ELISA (AntibodyShop, Grusbakken, Denmark), and LFABP
was measured using commercially available ELISA kits (CMIC Co., Tokyo, Japan) per the manufacturer’s instructions. The urine
KIM-1 ELISA was constructed using commercially available reagents (R&D Systems, Minneapolis, MN). eGFR was calculated using
the Chronic Kidney Disease Epidemiology Collaboration equation (14).

Statistical considerations

Sample size for this study was based on estimates of the difference in change in 24-h urine albumin excretion (the primary
outcome variable) and in eGFR. For urine protein excretion, we anticipated that a sample size of 30 participants would result
in 80% power to detect a difference between the n-3 PUFA and placebo periods of 10% at an α of 0.05.

The primary statistical analysis was the comparison of the difference in change in markers of kidney injury between the n-3
PUFA supplementation period and placebo period. The primary variable was the mean change in outcome variables from baseline
using repeated-measures analysis (i.e., generalized estimating equations with an exchangeable correlation structure). The
distributions of data were checked for normality and transformed where appropriate. Other outcome variables were analyzed
with a similar approach. Carryover effects of the n-3 PUFA supplement on the erythrocyte membrane fatty acids were evaluated
using treatment by assignment-order interaction terms.

We used interaction terms to evaluate effect modification by prerandomization medication use, namely, diuretics, statins,
glucose-lowering medications, and medications that block the RAAS (e.g., ACE inhibitors and angiotensin receptor blockers
[ARBs]). Statistical significance was P < 0.05. All analyses were performed using STATA version 11.1 (Stata Corporation, College Station, TX).

RESULTS

Study participants

Of 132 prescreened participants, we randomized 31 participants (Fig. 1). A lack of detectable urine dipstick proteinuria at screening was the most common reason for participants being ineligible.
One randomized participant withdrew before data collection, and one completed only one period. Baseline characteristics are
presented in Table 1, after excluding the participant who withdrew prior to data collection. Mean (SD) age of participants was 67.4 (11.5) years,
55% were male, and 61% were African American. Mean (SD) BMI was 31.6 (5.3) kg/m2. At baseline, 68% of participants had microalbuminuria (30–300 mg/day), and 26% had macroalbuminuria (>300 mg/day). Most
participants were taking lipid-lowering therapy (65%), RAAS inhibitors (68%), and/or hypoglycemic medications (81%).

Urine markers of kidney injury

Baseline values and changes in urine markers of kidney injury are reported in Table 2. Compared with the placebo period, n-3 PUFA had no significant effects on the 24-h urine excretion of albumin but significantly
lowered NGAL (−16%; 95% CI −29.1 to −0.5%; P = 0.04). Although n-3 PUFA did not significantly lower other markers of kidney injury, there was a consistent trend of reduction
during the n-3 PUFA supplementation period (Fig. 2).

Mean percent difference in 24-h urinary excretion of KIM-1, NGAL, LFABP, NAG, and albumin (ALB) comparing the n-3 PUFA to
the control period. (A high-quality color representation of this figure is available in the online issue.)

In stratified analysis by use of chronic stable doses of diuretics, statins, oral hypoglycemic medications, and RAAS-inhibitor
medications, interaction terms used to evaluate the effects of RAAS and n-3 PUFA supplementation use were significant for
NGAL (P = 0.01), NAG (P < 0.01), and microalbuminuria (P = 0.04), borderline significant for LFABP (P = 0.06), and nonsignificant for KIM-1 (P = 0.35) (Table 3). Within the stratum of RAAS-inhibitor users, there were significant reductions in all of the urinary kidney injury markers
except KIM-1. In contrast, among those not taking RAAS inhibitors, we observed nonsignificant increases in most markers and
a significant increase in NAG (1.32 units/day; 95% CI 1.02–1.70). The other medication classes did not demonstrate a similar
consistent pattern of effect modification (Fig. 3).

Mean percent difference in 24-h urinary excretion of KIM-1, NGAL, LFABP, NAG, and albumin (ALB) comparing the n-3 PUFA to
the control period, stratified by diuretic use (yes/no), statin use (yes/no), diabetes medication use (yes/no), and RAAS-inhibitor
(ACE/ARB) use (yes/no). (A high-quality color representation of this figure is available in the online issue.)

Compliance and safety

Analyses of erythrocyte membrane fatty acids (Table 4) revealed significant increases in EPA, DHA, and total n-3 fatty acids during the n-3 PUFA intervention period compared with
placebo, suggesting compliance with the intervention protocols. Carryover effects were statistically significantly for erythrocyte
membrane levels of EPA (P = 0.05), but not of DHA (P = 0.12) or total n-3 fatty acids (P = 0.08). Specifically, EPA levels in erythrocytes did not return to baseline in those first assigned to the n-3 PUFA.

A summary of adverse effects is presented in Table 5. Overall, adverse effects were infrequently reported, and there were no significant differences between treatment groups.
Furthermore, when participants were asked at the end of each period to identify the type of capsules that they believed they
were taking, 48% (n = 14) of participants taking placebo thought they were taking n-3 PUFA, whereas 47% (n = 14) of participants taking n-3 PUFA thought they were taking placebo, and 4% (n = 1) did not report treatment assignment (χ2, P = 0.3).

The effects of n-3 PUFA supplements on urine protein excretion have been tested in trials of primarily nondiabetic participants
with diverse underlying causes of CKD. In a meta-analysis of 17 trials, we reported a 19% reduction (95% CI −34 to −4; P = 0.01) in urine protein excretion with n-3 PUFA across all trials (20). A similar magnitude of effect was noted in the subgroup of trials (n = 7) that enrolled participants with diabetic kidney disease (−21% [−46 to 4]; P = 0.10).

We can only speculate on mechanisms that may account for the beneficial effects of n-3 PUFA supplementation on kidney-injury
processes. Effects on vascular function and renal hemodynamics are possible (4,21). A systematic review of fish oil supplements in hypertensive patients (five trials) reported that an average of 3 g/day
of n-3 PUFA reduced SBP/DBP by 5.5/3.5 mmHg (22). Our trial demonstrated a similar reduction in SBP, which could explain reductions in proteinuria and other markers of kidney
injury via hemodynamic effects. However, in spite of small differences in BP between the n-3 PUFA and placebo periods, we
did not detect an effect of n-3 PUFA supplementation on β2-microglobulin, cystatin C, or creatinine-based eGFR. These results suggest that n-3 PUFA effects on kidney injury markers
are likely independent of hemodynamic effects that effect GFR.

n-3 PUFA supplementation may also alter the balance of n-3 and n-6 fatty acids available for prostaglandin production, causing
a favorable shift toward production of prostaglandins with more vasodilator effects, enhanced immune function, and reduced
inflammation (16,23), mechanisms that may contribute to CKD progression in diabetic kidney disease. n-3 PUFA supplementation may also decrease
renal ischemic effects through rheostatic changes on erythrocyte membranes. Stirban et al. (21) noted improved postprandial microvascular reactive hyperemia after ischemia in adults with non–insulin-dependent diabetes
with 2 g/day of n-3 PUFA. This mechanism was believed to be largely independent of nitric oxide–mediated regulations of vascular
tone and more related to the influence of n-3 PUFA on prostaglandin pathways.

In our trial, n-3 PUFA had nonsignificant effects on urine albumin excretion (−7.2%; 95% CI −20.6 to 8.5; P = 0.35). The nonsignificant overall effects of n-3 PUFA supplementation on the primary outcome variable of urine albumin
excretion suggest a lack of benefit or reflect a trial is underpowered. Nonetheless, in subgroup analyses, n-3 PUFA significantly
reduced urine albumin excretion only in participants on RAAS-inhibition therapy, representing 70% of participants in this
trial. Indeed, the use of RAAS-inhibition therapy would be considered a standard of care for patients with diabetes and evidence
of kidney disease (2). A priori, one would expect that RAAS inhibitors would have had considerable antiproteinuric effects, limiting the ability
to detect the effects of additional agents. In our trial, the opposite occurred. For the most part, previous trials did not
report whether the antiproteinuric effects of n-3 PUFA were affected by the concurrent use of RAAS inhibition, but similar
results were reported in a trial of patients with immunoglobulin A nephropathy treated with RAAS. In this trial, enhanced
antiproteinuric effects were noted with the addition of 3 g/day of n-3 PUFA compared with RAAS treatment alone (24). Possible causes of additional antiproteinuric effects may include anti-inflammatory effects, reduced blood viscosity, and
increased erythrocyte flexibility leading to reduced renal tubular ischemia (5,25). The potential interaction between RAAS inhibitors and n-3 PUFA may have important clinical implications and needs to be
further investigated as a potential renoprotective combination in patients with diabetes and proteinuria.

Our trial has several strengths. First, the crossover design and relatively short-term intervention periods allowed for assessments
of the acute effects on urine albumin excretion and eGFR in the setting of stable doses of medications that may affect urine
protein excretion. Second, we enrolled diabetic participants with proteinuria, a common complication and early sign of CKD
in patients with diabetes. Third, the diabetes, hyperlipidemia (if present), and hypertension were treated. Hence, supplementation
with n-3 PUFA may have added benefit to conventional therapies in those with diabetes and early CKD. Fourth, the high rates
of adherence and follow-up, the use of 24-h urine sample collections, and the use of high-quality laboratory methods enhance
the internal validity of the trial. Fifth, novel markers of kidney injury are not susceptible to hemodynamic changes that
may alter urine albumin excretion.

There are also several limitations. We used a short-term crossover trial design, selected to enhance power to detect differences.
Although no carryover effects were detected in the analyses of outcome variables, we detected a carryover effect of the intervention
on EPA levels in erythrocytes. Erythrocyte EPA levels did not return to baseline at the end of the study in the group first
assigned to n-3 PUFA despite a 2-week washout period and a 6-week period on placebo. We note that this type of carryover effect
will tend to mask the effects of n-3 PUFA in the crossover analysis. Hence, future short-term trials of n-3 PUFA would benefit
from a longer washout period or a parallel-arm design. Another limitation pertains to our reporting a large number of secondary
outcomes and subgroup analyses stratified by baseline medication use. With this approach, one must be cautious in interpreting
statistical significance due to multiple comparisons and the risk of type 1 errors. For example, although we report significant
reductions in urine excretion of albumin, NAG, NGAL, and LFABP in those participants on RAAS therapy at baseline, there was
evidence of increased urine excretion of NAG in those not taking RAAS therapy. This finding can either reflect the truth or,
because of type 1 error associated with post hoc subgroup analyses, may be inappropriate. However, we feel that subgroup reporting
from this small trial is both informative and clinically relevant and may be helpful in the design of future trials of n-3
PUFA on kidney injury.

Each intervention capsule was a highly concentrated source of DHA/EPA derived from marine fish oils, and the dose was 4.0
g/day. This high dose of n-3 PUFA is far above the daily average intake of U.S. adults (0.15 g/day) (26) and contributes minimally to total calorie intake (∼40 kcal/day). This dose has therapeutic efficacy for triglyceride reduction
(∼25–40%) and is the dose currently sold by prescription for the treatment of hypertriglyceridemia. A lower dose of n-3 PUFA
(1.0 g/day) has reduced cardiovascular events in some (27,28) but not all (29) clinical trials. In our prior meta-analysis of n-3 PUFA supplementation on urine protein excretion, the dose of n-3 PUFA
did not modify the findings, but few trials used doses ≤1 g/day. Since 1 g/day is the currently recommended daily dose of
EPA/DHA for adults with coronary heart disease (30), further research is also needed to evaluate the effects of n-3 PUFA across a wider range of doses. Within the Vitamin D
and Omega-3 Trial, a National Institutes of Health–funded trial, 1,500 Vitamin D and Omega-3 Trial participants with diabetes,
randomly assigned in a 2 × 2 factorial design to vitamin D3 (cholecalciferol) 1,600 IU daily versus placebo and to EPA 500
mg plus DHA 500 mg daily versus placebo, will be studied to ascertain effects of study interventions on albuminuria and GFR
(31). Their trial is expected to finish in the year 2015.

Dose effects are also relevant to the observed adverse effects of n-3 PUFA supplements on fasting serum glucose. Increased
fasting glucose with n-3 PUFA supplementation has been reported in many, but not all, trials. A recent Cochrane meta-analysis
of 15 trials of fish oil supplementation on glucose homeostasis reported a nonsignificant pooled increase in fasting glucose
of 2.9 mg/dL (95% CI −2.3 to 8.3 mg/dL), with no effects on HbA1c (32). The mechanisms involved in increasing fasting glucose are unclear, but n-3 PUFA reduce hepatic synthesis of triglycerides
and increase hepatic fatty acid β-oxidation, resulting in increased hepatic glucose output through increased glycogenolysis
and/or gluconeogenesis (33). Nonetheless, whether a lower dose of n-3 PUFA is sufficient to lower kidney injury but below a threshold to cause hyperglycemic
effects needs to be tested.

In conclusion, in this randomized, controlled clinical trial, n-3 PUFA failed to reduce the primary outcome of urine albumin
excretion. However, there was a consistent trend of benefit for all urine biomarkers and a significant reduction in NGAL.
Furthermore, our post hoc subgroup findings raise the intriguing possibility that the greatest effects of n-3 PUFA are in
individuals on RAAS inhibitors. There were small reductions in SBP and DBP, an increase in fasting glucose, but no apparent
effect on eGFR with n-3 PUFA supplementation. Our results in the context of prior studies provide a strong rationale for larger
trials that are adequately powered for smaller effects on urine albumin excretion or trials that are conducted in participants
who take ACE/ARB therapy. In long-term trials of n-3 PUFA with clinical outcomes such as CKD progression, which are underway,
careful consideration should be given for the potential beneficial interactions between RAAS therapy and n-3 PUFA.

Acknowledgments

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R21-DK-080372 awarded to
E.R.M. Lovaza (n-3 PUFA) and placebo were kindly provided by GlaxoSmithKline. No other potential conflicts of interest relevant
to this article were reported.

E.R.M. analyzed and interpreted data, conceptualized and designed the study, and drafted the manuscript. S.P.J. analyzed and
interpreted data and drafted the manuscript. C.A.A., E.G., K.H.-R., S.T., and M.R.B. drafted the manuscript. J.C. and L.J.A.
conceptualized and designed the study and drafted the manuscript. E.R.M. is the guarantor of this work and, as such, had full
access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

This study was presented in abstract form at the American Society of Nephrology Kidney Week, San Diego, California, 30 October–4
November 2012.

. Management of hyperglycaemia in type 2 diabetes: a patient-centered approach. Position statement of the American Diabetes
Association (ADA) and the European Association for the Study of Diabetes (EASD). Diabetologia2012;55:1577–1596pmid:22526604